PUBLICIDAD EXTERIOR

The aims of this thesis were to analyse cellular and molecular aspects of Hmb

development in two vertebrate limb mutants, hypodactyly {Hd) in the mouse and talpid^ {ta in the chick. These mutants have an apparent disruption of normal limb patterning, such that there is severe loss of digits in hypodactyly and gain of digits in talpid^. It was hoped that by studying limb development in these mutants, it would be possible to make direct comparisons between normal and abnormal development, and by doing so, gain a better understanding of the nature of these defects and further unravel the sequence of events, both cellular and molecular, that result in patterning of the normal limb.

A t the time this study was commenced, very little work had been carried out on hypodactyly. The only information available was from the initial brief reports of Hummel (1963, 1970), which documented the skeletal defects observed in the limbs of adult mice. Heterozygotes {Hd/-¥) were reported to have a reduced digit 1 on the hindlimbs, whereas homozygotes {Hd/Hd) have a single digit on aU four limbs. The work described in the first section of Chapter 3 of this thesis, was carried out to confirm the findings of Hummel (1963, 1970) in regards to the morphology of the adult phenotypes. No previous studies have examined the morphology of H d mutant mice throughout the stages of embryogenesis when the limbs are forming. Therefore, in order to discover potential clues as to what the defects are and how they arise, it was of vital importance to carry out an embryological study in order to analyse the morphology of embryonic mutant limbs and pinpoint the time at which the defects are first visible.

The polarising region and apical ectodermal ridge are required for outgrowth and patterning along the antero-posterior and proximo-distal axes of the limb, and the

endogenous polarising region and ridge signals involved are proposed to be Shh and Fgf4. One could predict that the loss of digits in hypodactyly might be attributable to the loss or reduction of expression of Shh and/or Fgf4 during the stages of development when the hands and feet are being specified. Chapter 3 also addresses the signalling ability of posterior Hd/Hd limb mesenchyme, and the expression of Shh and Fgf4 in this m utant

The H d gene maps to murine chromosome 6 (Mock el al., 1987), and very recently the molecular basis of Hd has been identified as a deletion in the H oxal3 gene (Mortlock et al., 1996). Hox genes of paralogous group 13 are though to be involved in specification of the distal-most regions of the limbs; the hands and feet (Fromental- Ramain et al., 1996). The results described here add further evidence to support this possible role of H oxal3 in specification of hands and feet However, inactivation of H oxal3 in the H oxal3 knockout mouse, does not produce a comparable phenotype to that of Hd/Hd mice, instead Hd/Hd mice more closely resemble Hoxal3'^lHoxdl3'^' double knockout mice. These findings suggested the possibility \hd^,Hoxdl3 expression in the limbs of Hd/Hd mice may be altered, and so the final section of Chapter 3 analyses the expression of H oxdl3 and another Hoxd gene, H o x d ll. Knowing that Hd is caused by a mutation in H oxal3, makes this mutant a valuable tool for analysis of the

downstream targets and functions of the //ox genes during vertebrate limb development Chapter 4 discusses alterations in cell behaviour and additional morphological defects in the limbs of H d mutant mice, and discusses the findings in relation to their possible contribution to the final phenotypic outcome of the mutation.

The chicken mutant Talpid^ {ta^), has multiple defects that are widespread throughout the embryo (Ede and Kelly, 1964a, 1964b). Limbs of ta^/ta^ embryos are polydactylous, with up to 8-10 morphologically similar digits, many of which are partially fused and form a pattern which lacks any obvious antero-posterior polarity (Ede and Kelly, 1964b; Hinchliffe and Ede, 1967; reviewed by Hinchliffe and Johnson, 1980). The limb phenotype in ta^/ta^ embryos is associated with several different defects in the limb: there is a broadening of the developing limb buds; a lack of mesenchymal cell death and a defect in the mechanism that establishes antero-posterior polarity in the limbs (Ede and Kelly, 1964b; Hinchliffe and Ede, 1967; reviewed by Hinchliffe and Johnson, 1980; Izpisua-Belmonte et al., 1992).

The absence of antero-posterior polarity in the digit pattern of ta^/ta^ limbs is associated with a lack of discrete patterns of Hoxd gene expression in the developing limbs, which could account for the lack of specific digit identity (Izpisua-Belmonte et al.,

1992). It is not known how this altered pattern of Hoxd expression arises, but it could be

be due to a change in either signal distribution in the bud or the response of ta^/ta^ limb cells to polarising region signals (Izpisua-Belmonte et al., 1992). The work on Talpid^ described in Chapter 5 investigates different possible causes of the lack of antero­ posterior polarity in the limbs of ta^/ta^ embryos by mapping the distribution of

polarising activity in ta^/ta^ limbs , and analysing the response of ta^/ta^ limb cells to a polarising signal. In addition, it documents the expression patterns of other genes known to be involved in signalling and response in the developing hmb. Some of the work described in Chapter 5 of this thesis was carried out in collaboration with Dr. P. Francis- West and Dr. J-C. Izpistia-Belmonte.

Chapter 2

Materials and Methods used in the analysis of the mouse mutant